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Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth.

Sancho M, Diani E, Beato M, Jordan A - PLoS Genet. (2008)

Bottom Line: Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down.Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing.On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type.

View Article: PubMed Central - PubMed

Affiliation: Centre de Regulació Genòmica, Barcelona, Spain.

ABSTRACT
At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.

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Variant-specific effects of H1 depletion in cell progression are not restricted to T47D cells.HeLa, 293T, MCF7 and MCF10A cells were infected with the inducible shRNA-expression system to inhibit expression of (A) H1.2 or (B) H1.4, and the efficacy of linker histone depletion was tested in Western blot with specific antibodies. 293T cells infected with the empty shRNA-expression vector are shown as control. The percentage of H1.2 (A) or H1.4 (B) depletion upon quantification by band densitometry is shown underneath. Coomassie staining of H1 preparation is shown for each H1.2 KD cell line, were depletion of the lower band corresponding to H1.2 in response to Dox can be observed. (C) Cell lines indicated infected with the inducible shRNA-expression system against H1.2 or H1.4 were treated for 6 days with Dox or left untreated and observed at the optical microscopy for the number and morphology of attached cells. (D) The effect of H1.2 or H1.4 depletion on MCF7 and MCF10A cell proliferation was measured by following by FACS the variation of the proportion between shRNA-infected (GFP-positive) and parental cells (GFP-negative) from the 1∶1 seeding ratio over indicated days in the presence of Dox as in Figure 2B. (E) H1.2 knocked-down cells grown in the presence of Dox for 6 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death.
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pgen-1000227-g004: Variant-specific effects of H1 depletion in cell progression are not restricted to T47D cells.HeLa, 293T, MCF7 and MCF10A cells were infected with the inducible shRNA-expression system to inhibit expression of (A) H1.2 or (B) H1.4, and the efficacy of linker histone depletion was tested in Western blot with specific antibodies. 293T cells infected with the empty shRNA-expression vector are shown as control. The percentage of H1.2 (A) or H1.4 (B) depletion upon quantification by band densitometry is shown underneath. Coomassie staining of H1 preparation is shown for each H1.2 KD cell line, were depletion of the lower band corresponding to H1.2 in response to Dox can be observed. (C) Cell lines indicated infected with the inducible shRNA-expression system against H1.2 or H1.4 were treated for 6 days with Dox or left untreated and observed at the optical microscopy for the number and morphology of attached cells. (D) The effect of H1.2 or H1.4 depletion on MCF7 and MCF10A cell proliferation was measured by following by FACS the variation of the proportion between shRNA-infected (GFP-positive) and parental cells (GFP-negative) from the 1∶1 seeding ratio over indicated days in the presence of Dox as in Figure 2B. (E) H1.2 knocked-down cells grown in the presence of Dox for 6 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death.

Mentions: In order to investigate whether the effect of H1.2 and H1.4 deletion on cell proliferation was specific to the breast cancer cell line T47D used or if it is a more extended phenotype, we introduced the inducible shRNA expression system in a different breast adenocarcinoma cell line (MCF7), a non-tumoral breast epithelial cell line (MCF10A), a cervical adenocarcinoma cell line (HeLa) and an embryonic kidney cell line (293T). Efficient H1.2 and H1.4 depletion in response to Dox treatment was obtained for all cell lines, ranging from 63 to 95% (Figure 4A and 4B). In vivo observation of cells by microscope after 6 days of Dox treatment indicated that H1.2 depletion caused a clear defect in MCF7 cell growth, but not on HeLa, 293T or MCF10A (Figure 4C). Interestingly, H1.2 depletion was more pronounced in MCF10A than in MCF-7.


Depletion of human histone H1 variants uncovers specific roles in gene expression and cell growth.

Sancho M, Diani E, Beato M, Jordan A - PLoS Genet. (2008)

Variant-specific effects of H1 depletion in cell progression are not restricted to T47D cells.HeLa, 293T, MCF7 and MCF10A cells were infected with the inducible shRNA-expression system to inhibit expression of (A) H1.2 or (B) H1.4, and the efficacy of linker histone depletion was tested in Western blot with specific antibodies. 293T cells infected with the empty shRNA-expression vector are shown as control. The percentage of H1.2 (A) or H1.4 (B) depletion upon quantification by band densitometry is shown underneath. Coomassie staining of H1 preparation is shown for each H1.2 KD cell line, were depletion of the lower band corresponding to H1.2 in response to Dox can be observed. (C) Cell lines indicated infected with the inducible shRNA-expression system against H1.2 or H1.4 were treated for 6 days with Dox or left untreated and observed at the optical microscopy for the number and morphology of attached cells. (D) The effect of H1.2 or H1.4 depletion on MCF7 and MCF10A cell proliferation was measured by following by FACS the variation of the proportion between shRNA-infected (GFP-positive) and parental cells (GFP-negative) from the 1∶1 seeding ratio over indicated days in the presence of Dox as in Figure 2B. (E) H1.2 knocked-down cells grown in the presence of Dox for 6 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2563032&req=5

pgen-1000227-g004: Variant-specific effects of H1 depletion in cell progression are not restricted to T47D cells.HeLa, 293T, MCF7 and MCF10A cells were infected with the inducible shRNA-expression system to inhibit expression of (A) H1.2 or (B) H1.4, and the efficacy of linker histone depletion was tested in Western blot with specific antibodies. 293T cells infected with the empty shRNA-expression vector are shown as control. The percentage of H1.2 (A) or H1.4 (B) depletion upon quantification by band densitometry is shown underneath. Coomassie staining of H1 preparation is shown for each H1.2 KD cell line, were depletion of the lower band corresponding to H1.2 in response to Dox can be observed. (C) Cell lines indicated infected with the inducible shRNA-expression system against H1.2 or H1.4 were treated for 6 days with Dox or left untreated and observed at the optical microscopy for the number and morphology of attached cells. (D) The effect of H1.2 or H1.4 depletion on MCF7 and MCF10A cell proliferation was measured by following by FACS the variation of the proportion between shRNA-infected (GFP-positive) and parental cells (GFP-negative) from the 1∶1 seeding ratio over indicated days in the presence of Dox as in Figure 2B. (E) H1.2 knocked-down cells grown in the presence of Dox for 6 days were analyzed by FACS after PI staining to measure the percentage of cells in subG1phase as indicative of cell death.
Mentions: In order to investigate whether the effect of H1.2 and H1.4 deletion on cell proliferation was specific to the breast cancer cell line T47D used or if it is a more extended phenotype, we introduced the inducible shRNA expression system in a different breast adenocarcinoma cell line (MCF7), a non-tumoral breast epithelial cell line (MCF10A), a cervical adenocarcinoma cell line (HeLa) and an embryonic kidney cell line (293T). Efficient H1.2 and H1.4 depletion in response to Dox treatment was obtained for all cell lines, ranging from 63 to 95% (Figure 4A and 4B). In vivo observation of cells by microscope after 6 days of Dox treatment indicated that H1.2 depletion caused a clear defect in MCF7 cell growth, but not on HeLa, 293T or MCF10A (Figure 4C). Interestingly, H1.2 depletion was more pronounced in MCF10A than in MCF-7.

Bottom Line: Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down.Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing.On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type.

View Article: PubMed Central - PubMed

Affiliation: Centre de Regulació Genòmica, Barcelona, Spain.

ABSTRACT
At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants.

Show MeSH
Related in: MedlinePlus